Throughout this application various publications are referred to in parentheses. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Babesiosis is an emerging tick-borne disease caused by protozoan parasites of the genus Babesia that infects red blood cells (1, 2). Babesia infection can range from asymptomatic in healthy, immunocompetent persons to severe, life threatening syndromes such as hemolytic anemia, thrombocytopenia, organ failure, and even death, in individuals at risk, i.e., the elderly, asplenic persons, and those with coincident Lyme disease (3). Although babesiosis usually resolves in 2 to 3 weeks in immunocompetent individuals, there are reports of individuals carrying a low-grade asymptomatic infection for more than 2 years.
Babesia parasites in nature usually are transmitted to humans and animals by Ixodid ticks. The Ixodid ticks also transmit Borrelia burgdorferi and Anaplasma phagocytophilum, the etiologic agents for Lyme disease and Anaplasmosis, respectively. The Babesia parasites also are transmissible via blood transfusion or congenitally (4-7). In recent years, reports of tick-borne and transfusion-associated babesiosis cases have increased in number and geographic distribution in the United States. Because babesiosis may be asymptomatic, blood donors may not realize that they are infected, which poses a risk to the blood supply in areas of high endemicity. Between 1979 and 2009 over 159 transfusion-related B. microti cases, including nine deaths, have been documented in the U.S. (6, 8). Transmission of the Babesia parasites by transfusion is of particular concern, since recipients are likely to be in suboptimal health and less able to mount an efficient immune response: this could lead to rapid development of high parasitemia and difficulty clearing the infection.
In response to its increasing public health threat, the Centers for Disease Control and Prevention (CDC) made babesiosis a nationally notifiable disease as of January 2011 and received 1,124 cases reported from 15 states in 2011. To date all babesiosis cases reported to CDC with species-level information are caused by infection of Babesia microti, which occur mainly in the Northeast from coastal regions to areas such as inland counties of the lower Hudson River Valley of New York State, and some upper Midwest states such as Minnesota and Wisconsin (9-11). Human babesiosis is less common in Europe, where it is most often caused by infection with Babesia divergens (12).
The national surveillance case definition for babesiosis developed jointly by CDC and the Council of State and Territorial Epidemiologists recommends to confirm a diagnosis by one of the following laboratory methods: i) identification of intraerythrocytic Babesia organisms by light microscopy in a Giemsa, Wright, or Wright-Giemsa-stained blood smear; or ii) detection of Babesia microti DNA in a whole blood specimen by polymerase chain reaction (PCR); or detection of Babesia spp. genomic sequences in a whole blood specimen by nucleic acid amplification; or iii) isolation of Babesia organisms from a whole blood specimen by animal inoculation. Among these, animal inoculation is not a practical option. Therefore, microscopic examination of blood smears is currently the gold standard assay for confirmation of Babesia infection. However, this assay requires specially trained personnel for analysis, and has a detection of limit of 10-100 parasites/microtiter, which limits its use in patients with low parasitemia (13). Also, the infecting Babesia species can only be identified to the genus level based on morphological criteria. Furthermore, Babesia parasites can be difficult to distinguish from the early trophozoite (ring form) of Plasmodium parasites, particularly P. falciparum, in which ring forms are often the only stage that is observed. Thus, improved laboratory assays are needed both for clinical diagnosis and patient management, and for blood donor screening to prevent transfusion-transmitted babesiosis. DNA amplification using PCR is recognized as the most sensitive and specific methods for confirmation of B. microti infection and blood donor screening (14-16). However, there are no U.S. Food and Drug Administration-approved tests for Babesia. 
Recently, the use of real-time PCR has been described for detection of B. microti in ticks and clinical specimens from patients suspected of having tick-borne and transfusion-associated babesiosis (17-21). In these reports, the analytical sensitivity of real-time PCR assays was assessed by spiking cloned plasmid DNA to negative patient samples, which may result in inaccurate results due to the potential of preferred amplification of small plasmid DNA fragments rather than target sequence from the complete genome of a parasite in the PCR reaction (22, 23). Moreover, there are very limited data available on their clinical utilization in diagnosis and monitoring of treatment in patients.
The present invention addresses these needs by providing a validated, real-time PCR assay for rapid and accurate detection of B. microti. 